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Int Endod J. 2014 Aug;47(8):756-68. doi: 10.1111/iej.12214. Epub 2014 Jan 9.

The effectiveness of enzymic irrigation in removing a nutrient-stressed endodontic multispecies biofilm.

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Department of Restorative Dentistry, King's College London, London, UK; Department of Microbiology, Dental Institute, King's College London, London, UK.



To establish a nutrient-stressed multispecies model biofilm and investigate the dynamics of biofilm killing and disruption by 1% trypsin and 1% proteinase K with or without ultrasonic activation.


Nutrient-stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown on hydroxyapatite discs and in prepared root canals of single-rooted teeth in modified fluid universal medium. The treatment groups included trypsin, proteinase K, 0.2% chlorhexidine gluconate and 1% sodium hypochlorite (NaOCl) (with and without ultrasonics). NaOCl and chlorhexidine were the positive controls and untreated group, and sterile saline was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture.


Nutrient stress in the multispecies biofilm was apparent as the medium pH became alkaline, glucose was absent, and serum proteins were degraded in the supernatant. The CLSM showed the percentage reduction in viable bacteria at the biofilm surface level due to nutrient starvation. On the disc model, trypsin and proteinase K were effective in killing bacteria; their aerobic viable counts were significantly lower (P < 0.01) than the negative control and chlorhexidine. NaOCl was the most effective agent (P < 0.001). In the tooth model, when compared to saline, trypsin with ultrasonics caused significant killing both aerobically and anaerobically (P < 0.05). Chlorhexidine (1.46 ± 0.42), trypsin (3.56 ± 1.18) and proteinase K (4.2 ± 1.01) with ultrasonics were significantly effective (P < 0.05) in reducing the substratum coverage as compared to saline with ultrasonics (12% ± 4.9).


Trypsin with ultrasonic activation has a biofilm killing and disrupting potential.


stressed biofilm model

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